This application is a national stage application of PCT/GB 98/00848, filed Mar. 20, 1998, which claims foreign priority to GB 97 05787.1 filed Mar. 20, 1997.
The present invention relates to dendroaspin-based chimeric molecules which have anticoagulant, antiplatelet and other activities. The invention also relates to nucleic acid molecules encoding these chimeric dendroaspin molecules, cloning and expression vectors comprising such nucleic acids and host cells transformed with expression vectors so as to provide recombinant chimeric multifunctional dendroaspin. The invention further relates to pharmaceutical compositions comprising chimeric dendroaspin for use in the prevention or treatment of disease associated with thrombus formation or platelet aggregation. The invention also further relates to the use of a dendroaspin scaffold in the design and generation of chimeric dendroaspin derivatives having inhibitory activity against integrin binding activity of platelets plus some further functionality such as an anticoagulant or antithrombotic action.
The role of blood coagulation is to provide an insoluble fibrin matrix for consolidation and stabilization of a haemostatic plug. Formation of a cross-linked fibrin clot results from a series of biochemical interactions involving a range of plasma proteins.
Acute vascular diseases, such as myocardial infarction, stroke, pulmonary embolism, deep vein thrombosis and peripheral arterial occlusion are caused by either partial or total occlusion of a blood vessel by a blood clot.
The formation of a blood clot within a blood vessel is termed thrombosis and is dependent upon platelet aggregation. In the context of blood vessel injury (such as that which might arise in surgical procedures) the interaction of blood platelets with the endothelial surface of injured blood vessels and with other platelets is a major factor in the course of development of clots or thrombi.
Various agents for preventing formation of blood clots are now available, such as aspirin, dipyridamole and filopidine. These products generally inhibit platelet activation and aggregation, or delay the process of blood coagulation but they have the potential side effect of causing prolonged bleeding. Moreover, the effect of such products can only be reversed by new platelets being formed or provided.
Platelet aggregation is dependent upon the binding of fibrinogen and other serum proteins to the glycoprotein receptor IIb/IIIa complex on the platelet plasma membrane. GP IIb/IIIa is a member of a large family of cell adhesion receptors known as integrins, many of which are known to recognize an Arg-Gly-Asp (RGD) tripeptide recognition sequence.
Heparin and low molecular weight heparins have been used widely to treat conditions, such as venous thromboembolism, in which thrombin activity is responsible for the development or expansion of a thrombus. Although effective, heparin produces many undesirable side effects, including haemorrhaging and thrombocytopenia. A more specific and less toxic anticoagulant is therefore required.
Direct thrombin inhibitors are available and examples of these are hirudin, hirugen and hirulog (the latter two being synthetic hirudin derivatives), PPACK (a synthetic tripeptide) and argatroban (an arginine derivative). The actions of these inhibitors are reviewed in Lefkovits J and Topol E J (1994), Circulation 90:1522-1536. Although in theory, the bleeding risk with direct thrombin inhibitors is lower than with other antithrombotics because of their mono-target specificity, absence of direct platelet effects, and short half-life, bleeding still remains as the most concerning adverse effect.
There are a range of other thrombin inhibitors which have been developed (listed in table 1 of Lefkovits J and Topol E J supra) but these have turned out to be just too toxic for clinical use.
Localized narrowing of an artery caused by atherosclerosis is a condition which can usually be remedied surgically by the technique of balloon angioplasty. The procedure is invasive and causes some tissue damage to the arterial wall which can result in thrombus formation. Extracellular proteins such as fibronectin in the arterial wall become exposed to blood in the artery. Platelets bind to the RGD motif of fibronectin via integrin receptors which in turn leads to platelet aggregation and the start of the cascade of clotting reactions. An agent which specifically inhibits platelet aggregation at the sites of damage and which also inhibits clotting at these sites is required. The agent should be non-toxic and free of undesirable side effects such as a risk of generalized bleeding.
Integrins are a family of cell surface receptors that mediate adhesion of cells to each other or to the extracellular matrix (Kieffer N and Philips D R (1990) Annu Rev Cell Biol 6: 329-357; Hynes R O (1992) Cell 69: 11-25; McEver R P (1992) Curr Opin Cell Biol 4: 840-849; Smyth S S et al (1993) Blood 81: 2827-2843; Giancotti F G and Mainiero F (1994) Biochim Biophys Acta 1198: 47-64). They are composed of noncovalently associated xcex1 and xcex2 transmembrane subunits. There exist 16 different xcex1 and xcex2 different xcex2 subunits that heterodimerize to produce about 20 different kinds of receptors (Clark E A and Brugge J S (1995) Science 268: 233-239). Among the integrins, the platelet membrane integrin xcex1IIbxcex23 is one of the best characterized. Upon cell activation, the xcex1IIbxcex23 integrin binds several glycoproteins, predominantly through the Arg-Gly-Asp (RGD) tripeptide sequence (Pierschbacher M D and Ruoslahti E (1984) supra; Plow E F et al (1987) Blood 70: 110-115; Pytela R et al (1986) Science 231: 1559-1562) present in fibrinogen (Nachman R L and Nachman L L K (1992) J Clin Invest 69: 263-269), fibronectin (Gardner J M and Hynes R O (1985) Cell 42: 439-448), von Willebrand factor (Ruggeri Z et al (1983) J Clin Invest 72: 1-12), vitronectin (Pytela R M et al (1985) Proc Natl Acad Sci USA 82: 5766-5770), and thrombospondin (Karczewski J et al (1989) J Biol Chem 264: 21322-21326). The nature of the interactions between these glycoprotein ligands and their integrin receptors is known to be complex, and conformational changes occur in both the receptor (Sims P J et al (1991) J Biol Chem 266: 7345-7352) and the ligand (Ugarova T et al (1995) Thromb Haemostasis 74: 253-257).
Recently, many proteins from a variety of snake venoms have been identified as potent inhibitors of platelet aggregation and integrin-dependent cell adhesion. The majority of these proteins which belong to the so-called xe2x80x9cdisintegrinxe2x80x9d family share a high level of sequence homology, are small (4-8 kDa), cysteine-rich and contain the sequence RGD (Gould R J et al (1990) Proc Soc Exp Biol Med 195: 168-171) or KGD (Scarborough R M et al (1991) J Biol Chem 266: 9359-9362). In addition to the disintegrin family, a number of non-disintegrin RGD proteins of similar inhibitory potency, high degree of disulfide bonding, and small size have been isolated from both the venoms of the Elapidae family of snakes (McDowell R S et al (1992) Biochemistry 31: 4766-4772; Williams J A et al (1992) Biochem Soc Trans 21: 73S) and from leech homogenates (Knapp A et al (1992) J Biol Chem 267: 24230-24234). All of these proteins are approximately 1000 times more potent inhibitors of the interactions of glycoprotein ligands with the integrin receptors than simple linear RGD peptides; a feature that is attributed to an optimally favourable conformation of the RGD motif held within the protein scaffold. The NMR structures of several inhibitors including kistrin (Adler M et al (1991) Science 253: 445-448; Adler M and Wagner G (1992) Biochemistry 31: 1031-1039; Adler M et al (1993) Biochemistry 32: 282-289), flavoridin (Senn H and Klaus W (1993) J Mol Biol 234: 907-925), echistatin (Saudek V et al (1991) Biochemistry 30: 7369-7372; Saudek V et al (1991) Eur J Biochem 202: 329-328; Cooke R M et al (1991) Eur J Biochem 202: 323-328; Cooke R M et al (1992) Protein Eng 5: 473-477), albolabrin (Jaseja M et al (1993) Eur J Biochem 218: 853-860), decorsin (Krezel A M et al (1994) Science 264: 1944-1947), and dendroaspin (Jaseja M et al (1994) Eur J Biochem 226: 861-868; Sutcliffe M J et al (1994) Nature Struct Biol 1: 802-807) have been reported, and the only common structural feature elucidated so far is the positioning of the RGD motif at the end of a solvent exposed loop, a characteristic of prime importance to their inhibitory action.
Recent studies have implied a role for the amino acids around the tripeptide RGD in regulating the ligand binding specificity shown by snake venom proteins. Scarborough R M et al (1993) J Biol Chem 268: 1058-1065 examined a range of disintegrins and observed that those containing RGDW were very effective at inhibiting the interactions of fibrinogen to purified xcex1IIbxcex23 but not of vitronectin and fibronectin to purified xcex1vxcex23 and xcex15xcex21, respectively, whereas the converse was true for disintegrins containing the sequence RGDNP. Other regions of amino acid sequence divergence may also be contributory (Scarborough et al (1993) supra).
Dendroaspin, a short chain neurotoxin analogue containing the RGD sequence, and the disintegrin kistrin, which show little overall sequence homology but have similar amino acids flanking the RGD sequence (PRGDMP), are both potent inhibitors of platelet adhesion to fibrinogen but poor antagonists of the binding of platelets to immobilized fibronectin (Lu X et al (1994) Biochem J 304: 929-936). In contrast, elegantin, which has 65% sequence homology to kistrin but markedly different amino acids around RGD (ARGDNP), preferentially inhibited platelet adhesion to fibronectin as opposed to fibrinogen and binds to an allosterically distinct site on the xcex1IIbxcex23 complex.
Smith J W et al (1995) Journal of Biological chemistry 270: 30486-30490 undertook protein xe2x80x9cloop graftingxe2x80x9d experiments to construct a variant of tissue-type plasminogen activator (t-PA) which bound platelet integrin xcex1IIbxcex23. Amino acids in a surface loop of the epidermal growth factor (EGF) domain of t-PA were replaced with residues from a complementarity-determining region (CDR) forming one CDR of a monoclonal antibody reactive against the adhesive integrin receptor xcex1IIbxcex23. The resulting variant of t-PA (loop-grafted-t-PA) bound xcex1IIbxcex23 with nanomolar affinity and had full activity to both synthetic and natural substrates. The effects and applicability of loop grafting are altogether unpredictable and uncertain.
The present inventors have now discovered that the dendroaspin scaffold lends itself to modification. When dendroaspin (including the RGD motif) is modified to incorporate further functional amino acid sequences for example active portions or motifs of agonists, antagonists or inhibitors of factors in the clotting cascade, the resulting molecules are particularly useful as anticoagulants and do not suffer from the drawbacks associated with existing anticoagulants.
In first aspect the present invention provides a hybrid polypeptide comprising a first amino acid sequence including the RGD motif and conferring dendroaspin activity and a further amino acid sequence conferring activity other than that of dendroaspin activity.
The invention also provides a hybrid polypeptide having integrin binding activity comprising a dendroaspin scaffold and a further non-dendroaspin amino acid sequence, preferably of different activity.
Advantageously, the molecules of the invention have an integrin binding activity which when administered in vivo results in the binding of the molecules to platelets thereby inhibiting the aggregation of the platelets, at sites of injury. Moreover, the non-wild-type dendroaspin domain provides secondary, optionally further functionality eg antithrombotic action, inhibiting cell migration and proliferation and regulating signal transduction. Molecules of the invention are therefore bi- or multifunctional in their activities against blood coagulation, particularly thrombus formation and arterial/venous wall thickening at the sites of injury. Polypeptides of the invention may have activities against leukocyte recruitment, immune system activation, tissue fibrosis and tumorigenesis.
The polypeptide of the invention may comprise at least two said further amino acid sequences, preferably the two said further sequences are the same.
The further amino acid sequence may comprise two or more amino acid sequence portions separated by at least one amino acid residue of dendroaspin. The two or more sequence portions may be transposed with respect to one another and to the linear order of amino acids in the native further amino acid sequence. In other words, the native order of the two or more amino acid sequence portions is altered although the actual sequence of each portion may not necessarily be altered.
The said further sequence may be selected from platelet derived growth factor (PDGF), glycoprotein (GP) IBxcex1, hirudin, thrombin, throinbomodulin (particularly the fifth EGF-like domain thereof), vascular epidermal growth factor (VEGF), transforming growth factor-xcex21 (TGFxcex21), basic fibroblast growth factor (bFGF), angiotensin II (Ang II), factor VIII and von willebrand factor (vWF).
In this way the molecules of the invention may be rendered multifunctional so that they are active against more than just platelet aggregation, for example another component in the clotting cascade (eg thrombin activity), or the intracellular signaling cascade (eg growth factor). The modified dendroaspins of the invention may he engineered so that the further amino acid sequence has integrin binding activity, hereby providing a dendroaspin based molecule with augmented integrin binding activity.
The polypeptide of the invention preferably comprises an amino acid sequence as shown in FIG. 3 (SEQ ID NO:4). Prior to inclusion of said further amino acid sequence the dendroaspin scaffold of the invention includes homologous molecules which may share about 50% amino acid sequence homology, preferably about 65%, more preferably about 75% and even more preferably about 85% homology with dendroaspin.
Excluding the nucleic acid sequence encoding said further amino acid sequence nucleic acid sequences encoding the polypeptide of the invention may share about 50% nucleotide sequence homology, preferably about 65%, more preferably about 75% and even more preferably about 85% homology with a dendroaspin nucleotide sequence.
The polypeptides of the invention may comprise a greater or lesser number of amino acid residues compared to the 59 amino acids of dendroaspin. For example, the molecules of the invention may comprise a number of amino acid residues in the range 45 to 159, preferably about 49 to 85, more preferably about 53 to 69, even more preferably about 57 to 61.
The further amino acid sequence is preferably incorporated into (a) loop I and/or loop II; (b) loop I and/or loop III; (c) loop II and/or loop III; or (d) loop I, loop I and loop III of the dendrcaspin scaffold. Loop I comprises amino acid residues 4-16, loop II residues. 23-36 and loop III residues 40-50. However, the further amino acids being incorporated may extend into or substitute regions external to the loops, ie residues 1-3, 17-22 and 37-39 such that residues of the non-loop regions are augmented or substituted for those of the further amino acid sequence or sequences being inserted.
The further amino acid residues are preferably incorporated into either loop I or loop II. In this way the RGD-containing loop III is unaltered and so the integrin binding function of dendroaspin is retained.
A further RGD motif may be introduced into the dendroaspin scaffold, preferably into loop I or loop II, thereby increasing dendroaspin activity.
A preferred location for the inserted further sequence is at a site in dendroaspin scaffold between amino acid residues: 4-16, 18-21, 23-36, or 52-59.
Each inserted further amino acid sequence or portion of a further amino acid sequence is preferably an amino acid sequence in the range 3-40 amino acid residues, more preferably 3-16, even more preferably 3-14 amino acid residues long. The start of the inserted further amino acid sequence may be at any one of amino acid residues 1-57 of the dendroaspin scaffold. The end of the inserted further amino acid sequence may be at any one of amino acid residues 3-59 of the dendroaspin scaffold.
When two further amino acid sequences are inserted into the dendroaspin scaffold then the linear distance between these is preferably in the range 1-35 amino acids, more preferably 1-14 amino acids. When more than two further amino acid sequences are inserted then there is preferably at least one native dendroaspin amino acid residue separating each further amino acid sequence.
The RGD-containing loop may be modified by insertion, deletion or substitution of one of more amino acid residues, preferably a maximum of 8 or a minimum of 1 amino acids can be modified within loop III of dendroaspin.
The RGD loop preferably has an amino acid sequence as shown in FIG. 3B (residues 40-50 of SEQ ID NO:4). An advantage of modifying the RGD loop region is that the integrin binding activity may be enhanced and become more specific for certain glycoprotein ligands. Also, if one or more of the xe2x80x9cforeignxe2x80x9d further amino acid sequences grafted into the dendroaspin scaffold has steric effects on the RGD motif then loop III around the RGD site can be modified to overcome any steric hindrance thereby restoring, perhaps enhancing RGD functionality.
Loop I and/or loop II may be modified by insertion, deletion or substitution of one or more amino acid residues. Any suitable number of amino acids can be inserted into the dendroaspin scaffold to give the desired bi- or multi-functional activity although a number of residues in the range 14 to 36 are preferred for insertion at one or more sites in the dendrcaspin scaffold.
Modification of the loops may become necessary if a xe2x80x9cforeignxe2x80x9d further amino acid sequence grafted into the dendroaspin scaffold has a steric hindrance effect either on another grafted domain or on the RGD-containing loop. Computer assisted molecular modelling using Insight II software (Molecular Simulations Inc) can be used to predict the structure of the xe2x80x9cloop graftedxe2x80x9d dendroaspins of this invention. In instances where steric effects between the loops may serve to cause loss of functionality, these effects can be xe2x80x9cdesigned outxe2x80x9d by modifying appropriate parts of the dendroaspin molecule in an appropriate way. Sometimes this may involve inserting a number of suitable amino acid residues to extend one or more of the loop structures.
Preferred modification includes the insertion of polyglycine into the loop or loops of the dendroaspin scaffold in order to extend them. Other modifications comprising repeat units of an amino acid residue or number of residues can be used. Computer modelling studies can be used to design the loop modifications needed in order to extend the loops of dendroaspin.
In the design of a bifunctional or multifunctional molecule in accordance with the invention, xe2x80x9cfine tuningxe2x80x9d of activity, stability or other desired biological or biochemical characteristic may be achieved by altering individual selected amino acid residues by way of substitution or deletion. Modification by an insertion of an amino acid residue or residues at a selected location is also within the scope of this xe2x80x9cfine tuningxe2x80x9d aspect of the invention. The site-directed mutagenesis techniques available for altering amino acid sequence at a particular site in the molecule will be well known to a person skilled in the art.
In second aspect the invention provides a nucleic acid molecule encoding a polypeptide as hereinbefore defined.
The nucleic acid may be linked operatively to a promoter and optionally to a nucleic acid sequence encoding a heterologous protein or peptide thereby to encode a fusion product. The promoter is preferably xcex2-D-isopropyl-thiogalactopyranoside (IPTG) inducible and the heterologous protein or peptide may be glutathione S-transferase (GST).
This aspect of the invention also includes a plasmid comprising a nucleic acid as hereinbefore defined. The plasmid is preferably pGEX-3X.
In third aspect the invention provides a host cell transformed with a plasmid as hereinbefore defined, preferably said host cell is E coli. 
The invention therefore also provides a cell culture comprising transformed host cells as hereinbefore defined.
In fourth aspect the invention provides a method of producing a polypeptide as hereinbefore defined comprising culturing a host cell as hereinbefore defined so as to express said polypeptide, extracting the polypeptide from the culture and purifying the same.
In fifth aspect, the invention provides a method of producing a multifunctional anticoagulant comprising the steps of:
a) constructing an expression vector comprising a nucleic acid sequence encoding a dendroaspin scaffold operatively linked to a promoter and optionally linked to nucleic acid encoding an heterologous protein for co-expression therewith.
b) modifying at least a portion of the nucleic acid sequence of the vector encoding the dendroaspin scaffold, excluding the RGD motif, by one or more of insertion, deletion or substitution of nucleic acid residues so that on expression the dendroaspin scaffold comprises a further amino acid sequence of activity other than that of dendroaspin activity.
c) transforming a host cell with the vector and causing the host cell to express the modified dendroaspin nucleic acid sequence.
The method preferably further comprises the steps of:
d) extracting the modified dendroaspin from a host cell culture,
e) purifying the modified dendroaspin from the cell culture extract, optionally including the step of cleaving the dendroaspin from a co-expressed heterologous protein.
The heterologous protein is preferably GST and the purification preferably involves affinity chromatography using glutathione Sepharose 4B contained in the GST purification modules followed by factor Xa-mediated cleavage of the modified dendroaspins from GST.
The invention therefore provides a polypeptide as hereinbefore defined obtainable by the method of producing a multifunctional anticoagulant as defined above.
In sixth aspect the invention provides a pharmaceutical composition comprising a therapeutically effective amount of a polypeptide as hereinbefore defined, optionally further comprising a pharmaceutically acceptable excipient or carrier. A multiplicity of polypeptides of the invention of different functionalities may be combined together in a pharmaceutically acceptable form so as to provide a desired treatment. A multiplicity of polypeptides of the invention of different functionalities may be combined together in a pharmaceutically acceptable form so as to provide a desired treatment.
The polypeptide of the invention is preferably formulated for intravenous injection or intravenous infusion although other methods of administration are possible, eg subcutaneous or intramuscular should it be desired to provide a slow release into the circulatory system of an individual. Also possible is the formulation of the polypeptide for use with implanted controlled release devices such as those used to administer growth hormone for example.
One formulation may comprise extravasated blood combined with a polypeptide of the invention at a concentration in the range 1 nM-60 xcexcM. This blood may be stored in ready to use form and provides an immediate and convenient supply of blood for transfusion in cases when clotting must be avoided such as during or immediately following surgical procedures.
In seventh aspect the invention provides a polypeptide as hereinbefore defined for use as a pharmaceutical.
In eighth aspect the invention provides for the use of a polypeptide as hereinbefore defined for the manufacture of a medicament for the treatment of disease associated with thrombosis; more particularly thrombosis, myocardial infarction, retinal neovascularization, endothelial injury, dysregulated apoptosis, abnormal cell migration, leukocyte recruitment, immune system activation, tissue fibrosis and tumorigenesis.
The invention also provides methods for the treatment of disease associated with thrombosis; more particularly thrombosis, myocardial infarction, retinal neovascularization, endothelial injury, dysregulated apoptosis, abnormal cell migration, leukocyte recruitment, immune system activation, tissue fibrosis and tumorigenesis. The methods comprise administering a therapeutically effective amount of a polypeptide as hereinbefore defined.